U.S. patent number 4,687,841 [Application Number 06/789,271] was granted by the patent office on 1987-08-18 for peptide hydroxamic acid derivatives.
This patent grant is currently assigned to Monsanto Company. Invention is credited to William McC. Moore, Curtis A. Spilburg.
United States Patent |
4,687,841 |
Spilburg , et al. |
August 18, 1987 |
Peptide hydroxamic acid derivatives
Abstract
Novel peptide hydroxamic acid derivatives having useful
collagenase inhibitory activity and capable of forming affinity
resins for the purification of vertebrate collagenase are defined
by the following structural formula: wherein R=H or N-protecting
group or agarose.
Inventors: |
Spilburg; Curtis A.
(Chesterfield, MO), Moore; William McC. (St. Charles,
MO) |
Assignee: |
Monsanto Company (St. Louis,
MO)
|
Family
ID: |
25147131 |
Appl.
No.: |
06/789,271 |
Filed: |
October 18, 1985 |
Current U.S.
Class: |
530/331; 530/810;
530/813 |
Current CPC
Class: |
C07K
5/0823 (20130101); Y02P 20/55 (20151101); Y10S
530/813 (20130101); Y10S 530/81 (20130101) |
Current International
Class: |
C07K
5/097 (20060101); C07K 5/00 (20060101); C07C
103/52 () |
Field of
Search: |
;530/331,810,813 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Masui et al. Biochem. Med. 17, 215-221 (1977). .
Nishino and Powers, Biochem. 17, 2846 (1978). .
Nishino and Powers, Biochem. 18, 4340-4347 (1979)..
|
Primary Examiner: Foelak; Morton
Assistant Examiner: Nutter; Nathan M.
Attorney, Agent or Firm: Meyer; Scott J. Williams, Jr.;
James W.
Claims
What is claimed is:
1. A peptide hydroxamic acid derivative having the following
structural formula:
wherein R=H or N-protecting group or agarose.
2. The peptide hydroxamic acid derivative of claim 1 in which R is
an N-protecting group.
3. The peptide hydroxamic acid derivative of claim 1 in which R is
agarose.
Description
BACKGROUND OF THE INVENTION
This invention relates to novel peptide hydoxamic acid derivatives
having useful collagenase inhibitory activity and capable of
forming affinity resins for the purification of vertebrate
collagenase.
Collagenase is a highly specific, neutral protease which cleaves
undenatured collagen at a point about three quarters the distance
from the amino terminal end. The enzyme plays a critical role in a
variety of normal and pathological states such as resorption of the
post-partum uterus, wound healing, rheumatoid arthritis and tumor
invasion. Thus, in arthritic and arthrosic diseases, synovial
collagenase plays a prominent role in the degradation of the main
macromolecules of cartilage, collagen and proteoglycans, since once
the collagen fibers of the cartilage are destroyed, joint
destruction is irreversible. Accordingly, a specific collagenase
inhibitor would be considered as a potential therapeutic agent for
use against cartilage destruction in rheumatic diseases. Methods
for purification of vertebrate collagenase are useful for the study
of the role of collagenase in these and other such pathological
conditions.
BRIEF DESCRIPTION OF THE INVENTION
In accordance with the present invention, certain novel peptide
hydroxamic acid derivatives have been found to have useful
collagenase inhibitory activity and are capable of forming affinity
resins for the purification of vertebrate collagenase. These
peptide derivatives are defined by the following structural
formula:
wherein R=H or N-protecting group or agarose.
In the peptide structures shown herein, the amino acid components
are designated by conventional abbreviations as follows:
______________________________________ Amino Acid Abbreviated
Designation ______________________________________ L-Alanine Ala
Glycine Gly L-Leucine Leu L-Methionine Met L-Phenylalanine Phe
______________________________________
The N-protecting group depicted as R in the above structural
formula can be any group which will block .alpha.-amino functions
and, preferably, is alkanoyl, aroyl and cycloalkanoyl. Most
preferably, the blocking group is acetyl, benzoyl, carbobenzyloxy
(Z) or t-butyloxycarbonyl (t-BOC).
For use as an affinity resin, R is preferably agarose. Agarose is a
naturally occurring linear polysaccharide of galactose and
3,6-anhydrogalactose. A large variety of agaroses and modified
agaroses are available commercially which can be used in accordance
with the invention.
The novel peptide hydroxamic acid derivatives of this invention
have been found to be effective inhibitors of collagenase from
human synovial cells and human skin fibroblast cells. Thus, the
N-protected R-Pro-Leu-Gly-NHOH is about ten times more active than
the close analog R-Leu-Leu-Gly-NHOH. It is also a much more potent
collagenase inhibitor than the analogous thiopeptolide
Ac-Pro-Leu-Gly-S-Leu-Leu-Gly-OC.sub.2 H.sub.5 described in
application Ser. No. 571,227, filed Jan. 16, 1984 and now U.S. Pat.
No. 4,569,907, and assigned to a common assignee. Moreover, it is
estimated to be about 50-100 times more active than a commercially
available collagenase inhibitor product (Zincov) which chemically
is 2-(N-Hydroxycarboxamido)-4-methylpentanoyl-L-Ala-glycine
amide.
When the N-protecting group (or blocking group) is removed (that
is, when R=H) and the peptide hydroxamic acid is covalently bound
to agarose, a highly effective affinity resin is obtained. The
affinity resin is useful for the purification of large quantities
of human collegenase. By use of the affinity resin, skin fibroblast
collagenase was purified over 500 fold in 76% yield using this
single purification step. When assayed by polyacrylamide gel
electrophoresis with sodium dodecyl sulfate (SDS) and visualized in
the presence of mercaptoethanol, active collagenase isolated by
this method consisted of two bands, a major species with molecular
weight 45,000 and a minor one with molecular weight 50,000. When
the affinity-purified material was passed over an Ultrogel AcA 44
gel exclusion column, a molecular weight of 45,000 was found,
indicating the absence of sulfhydryl-linked subunits. Ultrogel AcA
44 consists of spherical beads of agarose trapped within a
cross-linked polyacrylamide gel and is commercially available from
LKB, Bromma, Sweden.
The affinity column also was able to isolate collagenase from human
synovial cells. The major purification occurs at the affinity
column step, but an additional gel filtration step was occasionally
required to achieve complete purification. The molecular weight and
amino acid composition of this synovial enzyme agree closely with
those of the skin enzyme; however, in this case, only a single
species of molecular weight 45,000 was found.
DETAILED DESCRIPTION OF THE INVENTION
While the specification concludes with claims particularly pointing
out and distinctly claiming the subject matter regarded as forming
the present invention, it is believed that the invention will be
better understood from the following detailed description of
preferred embodiments taken in connection with the accompanying
drawings in which:
FIG. 1 is a plot of the inhibition of human synovial collagenase by
the peptide hydroxamic acid derivative in one embodiment of the
invention, at 3.g .mu.M collagen ( ), 1.3 .mu.M collagen ( ) and
0.7 .mu.M collagen ( ).
FIG. 2 shows the affinity chromatographic profile of human skin
collagenase using an affinity column of Pro-Leu-Gly-NHOH covalently
bound to agarose.
The peptide hydroxamic acid derivatives of this invention can be
prepared by classical methods of peptide synthesis followed by
conversion of the peptide to the hydroxamic acid derivative. Thus,
the tripeptide moiety can be prepared by a series of coupling
reactions in which the constituent amino acids are linked together
by peptide bonds in the desired sequence. Commercially available
N-protected-L-proline also can be coupled with the commercially
available dipeptide L-leucylglycine. Activated esters of
N-hydroxysuccinimide are useful in this peptide synthesis by
procedure described by Anderson et al., J. Amer. Chem. Soc. 86,
1839-1842 (1964). According to this procedure,
dicyclohexycarbodiimide (C.sub.6 H.sub.11 N.dbd.C.dbd.NC.sub.6
H.sub.11) (DCC) is used to form the activated ester. ##STR1##
Z-Pro-Leu-Gly is amalogously converted to the active ester and then
reacted with hydroxylamine to form Z-Pro-Leu-Gly-NHOH.
According to a preferred method, the peptide hydroxamic acid
derivatives of this invention can be prepared by the following
series of steps:
I. Form the N-hydroxysuccinimide ester of the
N-protected-L-proline, e.g., t-BOC-proline or Z-proline, by
reaction with N,N-dicyclohexylcarbodiimide and N-hydroxysuccinimide
in about equimolar proportions.
II. Couple the resulting N-hydroxysuccinimide ester by reaction
with the dipeptide L-leucylglycine in about equimolar
proportions.
III. React the resulting t-BOC- or Z-Pro-Leu-Gly with
N,N-dicyclohexylcarbodiimide and N-hydroxysuccinimide in about
equimolar proportions.
IV. React the resulting N-hydroxysuccinimide ester with
hydroxylamine to form the hydroxamic acid derivative of the
peptide.
The reactions with DCC and N-hydroxysuccinimide are preferably
carried out in dioxane solutions. The formed dicyclohexylurea (DCU)
can be removed by filtration and the solvent removed by stripping
under reduced pressure. The remaining desired activated esters can
be recovered by crystallization.
Reaction II is conveniently carried out by dissolving the activated
ester of t-BOC-Pro or Z-Pro in dioxane solvent and adding to an
aqueous solution of Leu-Gly containing NaHCO.sub.3. Following
completion of the reaction, the solvent is removed by acidifying
and stripping under reduced pressure.
The hydroxylamine solution for reaction IV is readily prepared by
dissolving solid hydroxylamine HCl in dimethylformamide (DMF)
solvent, adding about an equimolar amount of triethylamine and
filtering off the hydrochloride salt. The remaining solution is
then mixed with the solution of the active ester of the peptide
from reaction III to form a solution of the hydroxamic acid
derivative of the peptide. The resulting solution is then
neutralized and the solvent is removed by evaporation to leave the
desired solid peptide hydroxamic acid derivative.
The affinity resin of the peptide hydroxamic acid derivative of
this invention can be prepared by removing the t-BOC, Z or other
blocking group and then coupling the unblocked peptide hydroxamic
acid derivative to agarose. A preferred method comprises coupling
the unblocked Pro-Leu-Gly-NHOH to activated CH-Sepharose.RTM. 4B
which is commercially available from Pharmacia Fine Chemicals AB,
Uppsala, Sweden. This material provides a six-carbon spacer arm and
an active ester for spontaneous coupling via amino groups. The
initial step of unblocking can be readily carried out by treatment
with anhydrous HF.
The following examples will further illustrate the invention
although it will be understood that the invention is not limited to
these specific examples.
EXAMPLE 1
Preperation of N-Benzyloxycarbonyl-L-prolyl-L-leucylglycine
Hydroxamic Acid (Z-Pro-Leu-Gly-NHOH)
Compounds were characterized by amino acid analysis after acid
hydrolysis, TLC (Silica Gel 60 F-254; CHCl.sub.3 :CH.sub.3
OH::3:1), melting point, IR (1% KBr pellets) and extinction
coefficient of the corresponding Fe.sup.3+ complex at 540 m.mu. (50
.mu.l of 10 mg/ml solution in dimethylformamide added to 3.0 ml of
2% FeCl.sub.3 in 0.10N HCl).
N-Benzyloxycarbonyl-L-proline succinimide ester (Z-Pro-OSu) was
prepared as follows:
Z-Pro (7.5 gm, 0.03 mole) and N-Hydroxysuccinimide (3.45 gm, 0.03
mole) were dissolved in cold dioxane and dicyclohexylcarbodiimide
(DCC) (6.18 gm, 0.03 mole) was added with rapid stirring. The
mixture was stirred overnight at room temperature and the next day
the solid dicyclohexylurea (DCU) was filtered off. The solvent was
removed and the oil was recrystallized from isopropyl alcohol.
Crystals of Z-Pro-OSu formed with scratching. Yield 8.4 gm. Melting
point 86.5.degree.-87.5.degree. C.
Z-Pro-OSu (3.46 gm, 10 mmol) as prepared above was dissolved in
dioxane and added to an aqueous solution of Leu-Gly (1.88 gm, 10
mmol) containing sodium bicarbonate (1.68 gm, 20 mmol). The
solution was stirred overnight at room temperature, acidified to pH
2.0 and all the solvent was removed under reduced pressure. The
remaining solid Z-Pro-Leu-Gly was washed with water and
recrystallized from ethanol-water. Yield 3.2 gm. Melting point
165.5-166. In a clean, dry beaker, Z-Pro-Leu-Gly (2.72 gm, 6.5
mmol) and N-hydroxysuccinimide (0.98 gm, 6.5 mmol) were dissolved
in 25 ml dioxane, and dicyclohexylcarbodiimide (DCC) (1.34 gm, 6.5
mmol) was added with stirring. The solution was stirred overnight,
and the following day the resulting solid dicyclohexylurea (DCU)
was filtered off and the remaining solution was retained. A
separate solution of hydroxylamine (0.68 gm, 9.75 mmol) was
prepared by dissolving hydroxylamine HCl in 10 ml dimethylfomamide
(DMF) and adding triethylamine (1.34 ml, 9.75 mmol). After
filtering off the hydrochloride salt, the two solutions were mixed
and stirred overnight at room temperature. The next day the
solution was neutralized, the solvent was removed on the rotovap
and the solid Z-Pro-Leu-Gly-NHOH was recrystallized from
ethanol-water. Yield 1.3 gm. Melting point 141-143. R.sub.f =0.63.
Amino Acid Analysis: Gly 1.00; Leu 1.04; Pro 0.95.
.epsilon..sub.540 = 910.
EXAMPLE 2
Several other hydroxamic acid compounds were prepared for
comparison (Table I, below) with the Z-Pro-Leu-Gly-NHOH of Example
1. These hydroxamic acid compounds were synthesized by the
nucleophilic attack of hydroxylamine on either peptide methyl
esters or succinimide esters. Compounds were characterized by amino
acid analysis after acid hydrolysis, TLC (Silica Gel 60 F-254;
CHCl.sub.3 :CH.sub.3 OH::3:1), melting point, IR (1% KBr pellets)
and extinction coefficient of the corresponding Fe.sup.3+ complex
at 540 m.mu. (50 .mu.l of 10 mg/ml solution in DMF added to 3.0 ml
of 2% FeCl.sub.3 in 0.10N HCl).
Method A. In a clean, dry beaker, 1.6 gm (5 mmol) Z-Leu-Gly (Sigma)
and O.75 gm (5 mmol) N-hydroxysuccinimide were dissolved in 25 ml
dioxane, and 1.03 gm (5 mmol) DCC was added with stirring. The
solution was stirred overnight, and the following day the solid DCU
was filtered off. A solution of hydroxylamine was prepared by
dissolving 0.52 gm NH.sub.2 OH.HCl (7.5 mmol) in 10 ml DMF and
adding 1.03 ml (7.5 mmol) triethylamine. After filtering off the
hydrochloride salt, the two solutions were mixed and stirred
overnight at room temperature. The next day the solution was
neutralized, the solvent was removed on the rotovap and the
remaining solid was recrystallized from ethyl acetate-hexane. The
oil was collected and triturated with ether. Yield 500 mg. Melting
point 110-113. Amino acid analysis: Gly 1.00; Leu 1.03. R f=0.80.
.epsilon..sub.540 =890.
Method B. In a small beaker, 0.34 gm (4.8 mmol) NH.sub.2 OH.HCl was
dissolved in 0.75 ml H.sub.2 O and 0.60 ml ethanol. When all the
solid dissolved, the solution was placed in an ice bath, and 1 ml
of 10N KOH (10 mmol) was added dropwise with stirring. The ice was
removed and the mixture was added to a solution of 1.4 gm (4.8
mmol) Z-Ala-Gly-OMe (Sigma) in 30 ml of methanol. The solution was
stirred for one hour at room temperature and the pH lowered to
below 7 by the dropwise addition of concentrated HCl. The beaker
was cooled on ice, the salt was filtered off and then all the
solvent was removed on the rotovap. This solid was taken up in
water and extracted with ethyl acetate. The water layer was
evaporated on the rotovap and the solid was recrystallized from
ethyl acetate. Yield 400 mg. Melting point 144.5-146.5. Amino acid
analysis: Gly 1.00; Ala 0.98. R.sub.f =0.70, .epsilon..sub.540
=890.
Z-Gly-NHOH. This compound was prepared from Z-Gly (4.2 gm, 20 mmol)
using Method A. After the hydroxamic acid was neutralized with
acid, the solvent was removed and the solid was recrystallized from
hot water. Yield 2.2 gm. Melting point 120.5-122. R.sub.f =0.74,
.epsilon..sub.540 =940.
Z-Gly-Gly-NHOH. This compound was prepared from Z-Gly-Gly (2.66 gm,
10 mmol) using Method A and recrystallized from boiling water.
Yield 1.6 gm. Melting point 150-151.5. R.sub.f =0.58,
.epsilon..sub.540 =900.
Z-Phe-Gly-NHOH. Z-Phe (3.0 gm, 10 mmol) and N-hydroxysuccinimide
(1.15 gm, 10 mmol) were dissolved in 40 ml cold DMF, and DCC (2.06
gm, 10 mmol) was added with rapid stirring. The solution was
stirrred for one hour on ice and then overnight at room
temperature. The next day the solid DCU was filtered off and the
clear solution was added to an aqueous solution of glycine methyl
ester, prepared by dissolving the hydrochloride salt (1.78 gm, 11
mmol) in water containing NaHCO.sub.3 (1.85 gm, 22 mmol). The
mixture was stirred for two hours at room temperature, the pH was
adjusted to 2, the solvent was removed and the remaining solid was
taken up in ethyl acetate. The solution was extracted with water,
the organic layer was dried over MgSO.sub.4 and the peptide was
crystallized by adding hexane to the boiling ethyl acetate
solution. Yield 3.0 gm. Melting pt. 116.5-118. R.sub.f =0.94. Amino
acid analysis: Gly 1.00; Phe 1.01. The hydroxamate was prepared
from the ester (2.1 gm, 5 mmol) using Method B. The crude solid was
taken up in ethyl acetate and recrystallized from ethyl
acetate-hexane. Yield 1.0 gm. Melting point 148-150. R.sub.f =0.82.
Amino acid analysis: Gly 1.00; Phe 1.02. .epsilon..sub.540
=1013.
Z-Met-Gly-NHOH. This compound was prepared from Z-Met-Gly-OEt
(Sigma-2.76 gm, 7.5 mmol) using Method B. The crude solid was
extracted with ethyl acetate and recrystallized from ethyl acetate.
Yield 1.0 gm. Melting point 133-134. R.sub.f =0.76. Amino acid
analysis Gly 1.00; Met 0.73 (starting material Gly 1.00; Met 0.60).
.epsilon..sub.540 870.
Z-Leu-Leu-Gly-NHOH. Z-Leu (2.7 gm, 10 mmol) and
N-hydroxysuccinimide (1.15 gm, 10 mM) were dissolved in dioxane,
and DCC (2.06 gm, 10 mmol) was added with rapid stirring on ice.
The solution was stirred overnight at room temperature, and the
next day the solid DCU was filtered off. The clear solution was
added to an aqueous solution of Leu-Gly (Sigma-2.07 gm, 11 mmol)
prepared by adding the solid Leu-Gly to NaHCO.sub.3 (1.85 gm, 22
mmol) dissolved in water. The mixture was stirred for four hours at
room temperature, the solution was adjusted to pH 2 by adding
concentrated HCl and all the solvent was removed on the rotovap.
The gummy mass was dissolved in ethyl acetate, extracted with water
and the organic layer was dried over MgSO.sub.4. The peptide was
recrystallized from ethyl acetate-hexane; the oil which formed was
collected and crystallized by triturating with ether. Yield 2.1 gm.
Melting point 96.5-99.5. The hydroxamate was prepared from
Z-Leu-Leu-Gly (1.8 gm, 4.l mmol) using Method A. Yield 0.9 gm.
Melting point 113-116. Amino acid analysis: Gly 1.00; Leu 2.04.
R.sub.f =0.81. .epsilon..sub.540 =930.
EXAMPLE 3
Preparation of Collagenase Affinity Columnn
Five hundred milligrams of Z-Pro-Leu-Gly-NHOH as prepared in
Example 1 were treated with anhydrous HF to remove the
carbobenzyloxy blocking group. The unblocked peptide was dissolved
in water and extracted two times with chloroform, once with hexane
and the aqueous layer was lyophilized. Prolylleucylglycyl
hydroxamic acid was coupled to activated CH-Sepharose 4B according
to the manufacturer's recommended general procedure for coupling
Sepharose. (Pharmacia Fine Chemicals AB, Uppsala, Sweden).
Specifically, the freeze dried resin (15 gm) was swollen and washed
with 3 liters of 1 mM HCl on a sintered glass funnel to give 45 ml
of gel with a capacity of 5-7 .mu.mole/ml. The unblocked peptide
(180 mg) was dissolved in 45 ml of 0.10M sodium bicarbonate, pH
8.0, and mixed with the gel for 60 minutes at 23.degree. C. The
coupled gel was then washed with 0.10M Tris, 0.50M NaCl, pH 8.0,
alternating with 0.10M sodium acetate, 0.50M NaCl, pH 4.0, and
stored at 4 .degree. in 0.05M Tris, 0.50M NaCl, 0.01M CaCl.sub.2 ,
pH 7.5. A 0.50 ml aliquot of the gel was hydrolyzed in 6N HCl and
found to contain 2.5 .mu.mole each of Pro, Leu and Gly.
EXAMPLE 4
Collagenase Inhibition Tests
The peptide hydroxamic acid derivatives prepared according to
Examples 1 and 2, above were tested for their activity as
inhibitors of collagenase as follows:
.sup.14 C-Collagen. .sup.14 C-collagen was prepared by reductive
methylation of calf skin collagen at 4.degree. C. using .sup.14
C-formaldehyde and sodium borohydride. Calf skin collagen (Sigma)
was dissolved at 7.5 mg/ml in 60 ml of 0.10M acetic acid and
dialyzed at 4.degree. C. against 0.15M potassium phosphate, pH 7.6,
for eight hours followed by dialysis overnight against 0.4M NaCl.
The collagen solution was then adjusted to pH 9.0 by addition of
0.50M sodium borate and then 1 mCi of .sup.14 C-formaldehyde (10
mCi/mmol) was added. After one minute, 0.10M sodium borohydride
(660 .mu.l in 1.3 mM NaOH) was added in four aliquots, followed by
an additional aliquot (340 .mu.l) 30 minutes later. The solution
was then dialyzed exhaustively against 0.01M acetic acid,
centrifuged to remove particulates and stored frozen in 1 ml
aliquots. Specific activity was 1.3.times.10.sup.6 DPM/mg.
Unlabeled collagen was prepared in the same way, stored frozen and
mixed with labeled collagen at a ratio of 9 to 1. For assay, the
diluted .sup.14 C-collagen was dialyzed 6-8 hours at 4.degree. C.
against 0.15M potassium phosphate, pH 7.6, followed by dialysis
overnight against 0.4M sodium chloride. This solution was
centrifuged to remove any undissolved collagen and stored at
4.degree. C.
Enzyme Assays. Collagenase assays were carried out according to the
method of Terato et al., Biochim. Biophys. Acta 445, 753 (1976).
They were performed in 1.5 ml polypropylene microfuge tubes. Each
assay tube contained 50 .mu.l of .sup.14 c-collagen solution (4
mg/ml) and 50 .mu.l of 1.0M glucose, 0.10M Tris, 0.4M NaCl, 0.02 M
CaCl.sub.2, pH 7.5. This solution was incubated for ten minutes at
35.degree. C. and the reaction initiated by the addition of 100
.mu.l of enzyme solution. Those samples containing procollagenase
were first activated by incubating 100 .mu.l aliquots with 1-5
.mu.l of 10 mg/ml trypsin (in 1 mM HCl) for 20 minutes at
23.degree. C., followed by 20 .mu.l of 5 mg/ml soybean trypsin
inhibitor (in 0.05M Tris, 0.01M CaCl.sub.2, pH 7.5) to quench the
trypsin activity. The collagenase assay was terminated after 30
minutes at 35.degree. C. by the addition of 20 .mu.l of 0.08M
1,10-phenanthroline in 50% (v/v) dioxane and the incubation was
continued for one hour at 35.degree. C. to denature the collagen
digestion products. Each sample was cooled for 15 minutes at
23.degree. and 200 .mu.l of dioxane was added with vigorous
vortexing to precipitate uncleaved collagen. Following
centrifugation at 11,000 RPM, 350 .mu.l aliquots were added to 5.0
ml of Pico-Fluor 30 to determine radioactivity.
Collagenase Inhibition. Collagenase activities were measured in the
presence of the test compounds by adding an aliquot of the compound
(0-85 .mu.l) and buffer to 100 .mu.l of glucose-collagen solution
to give a total volume of 185 .mu.l, incubating at 35.degree. C.
for 10 minutes and then initiating the reaction with 15 .mu.l of
purified collagenase. The rest of the assay was performed as
described above. To determine the mechanism of inhibition, the
following equation was used [(Holmquist and Vallee, J. Biol. Chem.
249, 4601 (1974)] ##EQU1## where A.sub.o is the activity in the
absence of inhibitor and A.sub.E is the activity in the presence of
inhibitor.
SK Hepatoma Cells. One vial of frozen SK Hepatoma cells (American
Type Cell Culture HTB-52) was rapidly thawed in a 37.degree. C.
water bath, the contents were transferred to a 75 cm.sup.2 T-flask
and 25 ml of Dulbecco's modified Eagle's medium (DMEM) containing
10% fetal bovine serum (FBS) was added. The flask was incubated at
37.degree. C. in an atmosphere containing 6-8% CO.sub.2. After one
day, the media was changed to remove the last traces of the
freezing media and four days later the cells were washed with 25 ml
phosphate buffered saline (PBS) containing 0.02% EDTA
(ethylenediamine tetraacetate). Five ml of the same media were
added and the cells were removed with gentle tapping. Five 75
cm.sup.2 T-flasks were inoculated with 1 ml of cells and 25 ml of
DMEM containing 10% FBS were added. For suspension culture, a 500
ml spinner flask was inoculated with cells from five 75 cm.sup.2
T-flasks and sufficient DMEM containing 10% FBS was added to bring
the volume to 500 ml. The spinner was maintained in a CO.sub.2
incubator at 37.degree. C. with 6-8% CO.sub.2. The media was
harvested three times per week by pouring off 400-450 ml and
leaving 50-100 ml for reinoculation. Cells were removed by
centrifugation and the conditioned media was sterile filtered and
used to treat fibroblast and synovial cells.
Skin Fibroblast Cells. One vial of normal skin fibroblasts was
purchased (American Type Cell Culture CRL 1224), thawed at
37.degree. C. and transferred to a 150 cm.sup.2 T-flask, fifty ml
of DMEM containing 10% FBS were added and the cells incubated for
one week at 37.degree. C. under 5% CO.sub.2. The cells were then
split into three T-flasks. When the flasks became overgrown with
cells, normal media was replaced with SK Hepatoma conditioned media
and changed twice a week. This media was stored and used as the
starting material for collagenase isolation.
Isolation and Culture of Synovial Cells. Approximately 2.0 gms of
synovial tissue were obtained from a patient undergoing synovectomy
of the knee. All procedures were carried out in a laminar flow hood
under aseptic conditions. The tissue was placed in 250 ml McCoys 5A
(modified) medium containing 200 .mu.g/ml gentamicin and stored
overnight at 0.degree. C. The next day the tissue was warmed to
room temperature, cut into 0.25 cm pieces and added to 11 ml of
serum-free DMEM, containing 4 mg/ml clostridial collagenase
(Worthington). After incubating the mixture for one hour at room
temperature, an equal volume of 0.25% trypsin was added and the
incubation continued for an additional thirty minutes. The cells
were spun down at 400.times.g for ten minutes, resuspended in 20 ml
trypsin/EDTA (Gibco 10X) and incubated for 30 minutes with
occasional mixing by drawing through a 25 ml pipet. The suspension
was centrifuged and the pellet was washed two times with PBS:DMEM
(1:1) containing 10% FBS. The cells were resuspended at
1.times.10.sup.6 cells/ml in DMEM containing 10% FBS and 100 .mu.g
gentamicin. After incubating overnight at 37.degree. in a CO.sub.2
incubator 5-8% CO.sub.2), the non-adherent cells were aspirated off
and the adherent cells were washed with PBS:DMEM (1;1) containing
10% FBS, followed by DMEM containing only 10% FBS. At the first
passage, the original T-flask was split one to four. As described
above, when the cells became confluent, 30% SK Hepatoma conditioned
media was added to the T-flask to stimulate the cells to produce
collagenase. This was the starting material for enzyme isolation.
To determine the K.sub.I values of the peptide hydroxamic acid
derivatives tested for collagenase inhibition, the inhibition by
Z-Pro-Leu-Gly-NHOH was measured over a ten-fold concentration range
at three different substrate concentrations, 3.9 .mu.M, 1.3 .mu.M
and 0.7 .mu.M and the data plotted as a Dixon plot. All the plots
were linear and intersected on the abscissa, -K.sub.I, indicating
non-competitive inhibition as seen from FIG. 1. The other test
compounds were assumed to inhibit by the same mechanism so that all
studies were performed at only a single substrate concentration, 10
.mu.M, and the K.sub.I values were determined from the intersection
with the abscissa.
Table I, below, sets forth the K.sub.I values of these peptide
hydroxamic acid derivatives when thus tested for inhibition of
human collagenase as above isolated from skin fibroblasts and
synovial cells. With a K.sub.I of 4.times.10.sup.-5 M, the
Z-Pro-Lue-Gly-NHOH was found to be ten times more effective than
the corresponding Z-Leu-Leu-Gly-NHOH analog and, moreover, this was
the most effective inhibitor among the compounds tested. It is also
estimated to be about 50 to 100 times more active than Zincov which
has a K.sub.I of 2.times.10.sup.-3 M.
TABLE I ______________________________________ K.sub.I VALUES OF
PEPTIDE HYDROXAMIC ACID INHIBITORS OF COLLAGENASE.sup.a K.sub.I
(mM) SYNOVIAL FIBROBLAST COLLA- COLLA- TEST INHIBITOR GENASE GENASE
______________________________________ Z--Gly--NHOH 0.96 0.60
Gly--NHOH 18. >1 Z--Gly--Gly--NHOH 2.0 3.0 Z--Ala--Gly--NHOH 2.6
1.3 Z--Leu--Gly--NHOH 0.17 0.48 Z--Phe--Gly--NHOH 0.10 0.15
Z--Met--Gly--NHOH 0.20 0.15 Z--Leu--Leu--Gly--NHOH 0.33 0.30
Z--Pro--Leu--Gly--NHOH 0.047 0.040
______________________________________ .sup.a 0.50 M NaCl, 0.01 M
CaCl.sub.2, 0.05 M Tris, pH 7.5, 35.degree. C.
EXAMPLE 5
Purification of Collagenase
The Affinity matrix prepared by coupling Pro-Leu-Gly-NHOH to
agarose according to Example 3 was used to purify collagenase as
follows. In a typical test, 100 ml of SK Hepatoma media were
removed from either skin fibroblasts or synovial fibroblasts and
treated with ammonium sulfate to 55% of saturation. The precipitate
was collected by centrifugation, dissolved in water and then
dialyzed against 0.50M NaCl, 0.01M CaCl.sub.2, 0.01M Tris, pH 7.5.
Most cells produce collagenase as an inactive zymogen or as an
enzyme inhibitor complex so that no activity is observed in the
starting cell culture media. However, when this same media is
treated with trypsin, collagenase activity is generated. Therefore,
the dialyzed protein solution was made one mg/ml in trypsin,
allowed to stand at room temperature for twenty minutes and then
solid soybean trypsin inhibitor was added to give a final
concentration of 1.5 mg/ml. The mixture was then pumped onto the
affinity column (1.5.times.15 cm) at 15 ml/hr and the column was
washed with 0.50M NaCl, 0.01M CaCl.sub.2, 0.01M Tris, pH 7.5. As
shown in FIG. 2, most of the protein washed through the column and
all the activity was bound. When the protein absorbance returned to
zero, the column was washed with 0.50M NaCl, 0.10M CaCl.sub.2,
0.10M Tris, pH 9.0. Since collagenase has lower stability at high
pH, 2.5 ml fractions were collected into one ml of 0.50M NaCl,
0.60M Tris, pH 6.5. Under these conditions, all the activity was
eluted in a single protein peak (FIG. 2). For the skin enzyme, when
the three or four peak fractions were pooled and examined by
polyacrylamide gel electrophoresis with sodium dodecyl sulfate
(SDS), one major band and a faint minor band were found at
molecular weight 45,000 and 50,000 respectively, indicating that
the enzyme was essentially pure. For the synovial enzyme there were
still some impurities remaining after this affinity step so that an
additional gel exclusion column was required for complete
purification. An AcA 44 gel exclusion column was used for this
purpose. When the active fractions from this column were pooled and
examined on SDS gels, a single band of molecular weight 45,000 was
found. The purification scheme for both enzymes is summarized in
Table II and, as shown there, both can be isolated in high yield
and with high specific activity. Importantly, when either of these
enzymes was incubated with collagen and the cleavage products
visualized on SDS gels, only the characteristic 3/4-1/4 collagen
cleavage products were observed.
After each run, the column was regenerated by washing with 0.05M
EDTA, 0.50M NaCl, pH 7.5, followed by 0.50M NaCl, 0.01M CaCl.sub.2,
0.01M Tris, pH 7.5. One column was used over 25 times over a period
of six months, with no apparent loss of capacity or other
chromatographic properties.
Trypsin activation was essential for the isolation of either
enzyme. When media was treated with ammonium sulfate as described
above and then pumped onto the column with no trypsin activation,
no protein was eluted with pH 9.0 buffer. However, if each fraction
of the unbound material was activated with trypsin for 20 minutes
and this exogenous activity quenched with soybean inhibitor, all
the collagenase activity was found in the column wash through,
indicating that the inactive enzyme was not bound to the resin.
TABLE II
__________________________________________________________________________
PURIFICATION OF HUMAN COLLAGENASES SKIN SYNOVIAL Total Sp. Act.
Purifi- Total Sp. Act. Purifi- Protein Activity.sup.a .times.
(U/A.sub.280) .times. Recov- cation Protein Activity.sup.a .times.
(U/A.sub.280) Recovery cation Step (A.sub.280) 10.sup.-3 10.sup.-3
ery % (-fold) (A.sub.280) 10.sup.-3 10.sup.-3 % (-fold)
__________________________________________________________________________
Media 432 8450 20 100 1 3486 5890 1.7 100 1 Ammonium 92 6290 68 74
3.4 869 2051 2.4 35 1.4 Sulfate Affinity 0.63 6600 10600 76 530 1
3484 3484 59 2050 AcA 44 0.42 4200 10000 71 5880
__________________________________________________________________________
.sup.a Activity expressed as DPM/min.
Various other examples will be apparent to the person skilled in
the art after reading the present disclosure without departing from
the spirit and scope of the invention and it is intended that all
such examples be included within the scope of the appended
claims.
* * * * *